水飞蓟宾A与水飞蓟宾B在大鼠体内的药动学及生物利用度研究_386

水飞蓟宾A与水飞蓟宾B在大鼠体内的药动学及生物利用度研究_386 水飞蓟宾A与水飞蓟宾B在大鼠体内的药动学及生物利用度 研究 *褚扬，李伟，万梅绪，栗志文，郭嘉华，马晓慧，周水平 (天津天士力集团研究院药理毒理所，天津 300410) [摘要] 目的:建立一种简单、快速、灵敏的液-质联用法(LC-MS/MS)，同时 测定大鼠血浆中水飞蓟宾A与水飞蓟宾B浓度。方法:0.1 mL血浆样品经沉淀 蛋白预处理后，以甲醇-0.1%甲酸(48:52，v/v)为流动相，采用Zorbax Eclipse XDB-C 色谱柱(150 × 2.1 mm，5 μm)分离，以ESI源负离子模式、选择反应18 监测(SRM)方式进行定量分析。结果:水飞蓟宾A与水飞蓟宾B在血浆中的线性 -1-1范围为2.0~50000 ng?mL，最低定量限(LLOQ)为2.0 ng?mL。药物的日内、日 间精密度在9.1%以内，准确度(相对误差)小于?5.7%。结论:本方法成功地用 于水飞蓟宾A与水飞蓟宾B各自的药动学及生物利用度研究。 [关键词]水飞蓟宾A;水飞蓟宾B;液-质联用;药动学;生物利用度 [中国图分类号]R 969 [文献标识码]A Investigation of the bioavailability and pharmacokinetics of silibinin A and silibinin B in rats CHU Yang, LI Wei, WAN Mei-xu, LI Zhi-wen, GUO Jia-hua, MA Xiao-hui, ZHOU Shui-ping (Tasly R&D Institute, Tianjin Tasly Group Co., Ltd., Tianjin 300410, ) China [ABSTRACT] OBJECTIVE: To develop a simple, rapid and sensitive 作者简介: 褚扬(1982-)，男，硕士 研究方向: 药物代谢及药动学 Tel.: (022) 26736372 Fax: (022) 26736376 E-mail: chuyang0610@163.com - 1 - LC/MS/MS method for the determination of silibinin A and silibinin B in rat plasma. METHODS :Sample pretreatment involved in one-step protein precipitation with acetonitrile of 0.1 mL plasma. The separation of silibinin A and silibinin B was performed on a Zorbax Eclipse XDB-C column 18 (150 × 2.1 mm, 5 μm) with the mobile phase of methanol-water-formic acid (48:52:0.1, v/v/v). The analytes were monitored with negative electrospray ionization (ESI) by selected reaction monitoring (SRM) mode. RESULTS :The linearity of this method was found to be from 2.0 to 50000 -1-1ng?mL with a lower limit of quantitation (LLOQ) of 2.0 ng?mL for both silibinin A and silibinin B, respectively. The precision was less than 9.1% (intra- and inter-), and the accuracy (relative error) was within ?5.7%. CONCLUSION :This method was successfully applied to investigate the pharmacokinetics and bioavailability of silibinin A and silibinin B in rats, respectively. [KEY WORDS] Silibinin A; Silibinin B; LC/MS/MS; Pharmacokinetics; Bioavailability 水飞蓟宾是菊科植物水飞蓟中提取分离得到的黄酮类化合物，具有保肝、护 [1-4][5,6]肝等功效。近年来药效学研究明，水飞蓟宾还具有较强的抗炎，抗肿瘤 [7-13][14]活性。定天明等采用胶束电泳毛细管色谱法对水飞蓟宾的非对映体(水飞蓟 宾A和水飞蓟宾B)进行了分离，从而证明了水飞蓟宾为两个非对映体的混合物， 。 其化学结构见图1 [15-21]目前，已有多篇文献报道了水飞蓟宾在动物和人体内的定量分析研究， [22]但有关水飞蓟宾A和水飞蓟宾B各自的药动学研究报道极少。Rickling等采 用高效液相色谱-电化学法(HPLC-ECD)测定人血浆中水飞蓟宾A和水飞蓟宾B的 浓度，但该方法的灵敏度低，且样品预处理较为繁琐，不能满足药动学研究需要。 本实验建立了一种简单、快速、灵敏的LC-MS/MS方法，同时测定大鼠血浆 中水飞蓟宾A和水飞蓟宾B浓度，并成功用于水飞蓟宾A和水飞蓟宾B各自的药 动学及生物利用度研究。 - 2 - 图1 水飞蓟宾A (a)和水飞蓟宾B (b)的化学结构 Fig. 1 Chemical structures of silibinin A (a) and silibinin B (b) 1 材料与方法 1.1 药品与试剂 水飞蓟宾A和水飞蓟宾B品(天津天士力集团研究院自制，纯度>98.5%);内标柚皮苷(中国药品生物制品检定所，纯度>99.0%，批号:110722-200610);水飞蓟宾胶囊(天津天士力制药股份有限公司，规格:35 mg水飞蓟宾/粒，批号:090509);乙腈为色谱纯(美国Fisher公司);其余试剂均为分析纯。 1.2 仪器 Finnigan TSQ Quantum三重四极杆液-质联用仪(美国Finigan公司);XW-80A微型漩涡混合仪(上海卢西分析仪器厂有限公司);TG 16W微量高速离心机(长沙平凡仪器仪表有限公司)。 1.3 色谱条件 色谱柱:Zorbax Eclipse XDB-C柱(150 × 2.1 mm，5 μm)，美国Agilent18 -1公司;流动相:甲醇-0.1%甲酸(48:52，v/v);流速:0.25 mL?min;柱温:30?;进样量:10 µL。 1.4 质谱条件 离子源为电喷雾离子源，负离子方式检测;喷雾电压3800 v;毛细管温度390?;鞘气(N2)压力25 Psi;辅助气(N2)压力3 psi;碰撞气(Ar)压力为1.4 mTorr;扫描方式为选择性反应监测(SRM);碰撞能量(CE)分别为20 eV(水飞蓟宾A和水飞蓟宾B)和34 eV(柚皮苷)，用于定量分析的离子反应分别为m/z 481.1?m/z 300.9(水飞蓟宾A和水飞蓟宾B)和m/z 579.2?m/z 271.1 (柚皮苷)。水飞蓟宾A、水飞蓟宾B和内标柚皮苷的二级全扫描质谱图见图2。 图2 水飞蓟宾A (a)、水飞蓟宾B (b)和柚皮苷(c)的质谱图 Fig. 2 Product ion mass spectrum of silibinin A(a), silibinin B(b) and naringin(c) 1.5 实验动物与实验 1.5.1 实验动物 Wistar大鼠，雄性，体重(220?20) g (北京维通利华实验动物中心)。 1.5.2 静注、灌胃用水飞蓟宾溶液的配置 精密称取水飞蓟宾140 mg，溶于25 - 3 - mL聚乙二醇200:乙醇:二纯水(5:2:3，v/v/v)混合溶液中，即得水飞蓟宾静注溶液;分别取4，8和16粒水飞蓟宾胶囊，去壳，混悬于70 mL二纯水中，配置成水飞蓟宾低、中、高3个剂量的灌胃液。上述给药溶液均需现用现配。 -11.5.3 给药及血样采集 5只大鼠按28 mg?kg静脉注射给予水飞蓟宾静注溶 -1-1相当于水飞蓟宾A13.3 mg?kg，水飞蓟宾B14.7 mg?kg);15只大鼠分别按液( -128，56和112 mg?kg灌胃给予低、中、高3个剂浓度的水飞蓟宾混悬液(相当 -1-1于水飞蓟宾A13.3，26.6和53.2 mg?kg，水飞蓟宾B14.7，29.4和58.8 mg?kg)。大鼠给药后0，0.17，0.33，0.67，1.0，1.5, 2.0，3.0，4.0，6.0，8.0，12.0 -1h，经大鼠眼球后静脉丛采集静脉血约0.3 mL，置肝素化试管中，3500 r?min离心10 min，分离血浆，置–20?冰箱中冷冻保存待测。 1.6 血浆样品预处理 取血浆样品100 μL置于1.5 mL Eppendorf管中，依次加入乙腈100 μL， -1内标溶液(200 ng?mL柚皮苷溶液，乙腈配置)100 μL，旋涡1 min，然后于12000 -1r?min离心8 min，取上清液10 μL进行LC-MS/MS分析。 2 结果 2.1 专属性考察 由图3可见，在“1.3”所述条件下，水飞蓟宾A、水飞蓟宾B和内标的保留时间分别为6.45，7.35和2.55 min，且血浆中的内源性物质不干扰待测物与内标的测定。 图3 血浆样品色谱图 a-空白血浆;b-空白血浆加入水飞蓟宾A和内标;c-空白血浆加入水飞蓟宾B和内标;d-给药后的 血浆样品 Fig. 3 Chromatograms of sample in plasma a-blank plasma; b-blank plasma spiked with silibinin A and IS; c-blank plasma spiked with silibinin B and IS; d-plasma sample 2.2 线性范围及灵敏度 取大鼠空白血浆，加入水飞蓟宾A、水飞蓟宾B标准溶液，配制成含水飞蓟宾 -1A和水飞蓟宾B2，5，20，50，500，2000，20000和50000 ng?mL的血浆样品， - 4 - 按“1.6”项操作，记录色谱图;以血浆样品中待测物的浓度为横坐标，待测物 2与内标物的峰面积比值为纵坐标，用加权(w=1/X)最小二乘法进行回归运算，求得水飞蓟宾A的回归方程为:Y= 0.00125 X+0.00063 (r=0.9981)，水飞蓟宾B的回归方程为:Y = 0.00122 X+0.00096 (r=0.9959)，本法水飞蓟宾A和水飞蓟宾B -1-1的线性范围均为2~50000 ng?mL，LLOQ为2 ng?mL (S/N>10)。 2.3 精密度与准确度 取大鼠空白血浆若干份，分别加入水飞蓟宾A和水飞蓟宾B标准溶液，配置成 -1水飞蓟宾A和水飞蓟宾B浓度为20，500，20000 ng?mL的血浆样品，按“1.6” 样本分析，连续测定3天，计算精密度(日内、日间RSD)项操作，每个浓度进行6 与(RE)结果见表1。 表1 精密度与准确度实验结果 Tab. 1 Precision and accuracy of the method 2.4 提取回收率 取空白血浆100 μL，制备水飞蓟宾A和水飞蓟宾B浓度为20，500，20000 -1ng?mL的血浆样品，按“1.6”项操作，每个浓度进行3样本分析，记录色谱峰。同时，另取纯水100 μL代替空白血浆，同法操作，获得相应峰面积，以二者色谱图的峰面积之比考察样品的提取回收率。结果表明，在上述3个浓度下水飞蓟宾A的提取回收率分别为95.2?2.2%，93.2?1.7%和95.4?4.5%，水飞蓟宾B的提取回收率分别为94.3?5.1%，94.7?4.1%和96.1?1.9%，内标的提取回收率为93.8?5.4%。 2.5 稳定性考察 本实验分别考察了血浆样品,20:C冻存2个月，冻-融3个循环及血浆样品处理后室温放置24 h的稳定性。结果表明，水飞蓟宾A和水飞蓟宾B在上述3个条件下稳定性良好。 2.6 药动学及生物利用度研究 水飞蓟宾A和水飞蓟宾B在大鼠体内的药-时曲线图见图4，Topfit 2.0软件用于计算其主要药动学参数，见表2和表3。经统计分析，水飞蓟宾A和水飞蓟宾B的主要药动学参数C，AUC和AUC与给药剂量呈线性关系。由公式:F = (AUC max0-t0-i.g., - 5 - / AUC) × 100%，计算得到水飞蓟宾A和水飞蓟宾B的绝对生物利用度分别为i.v. 2.86%和1.93%。 图4 水飞蓟宾A和水飞蓟宾B在大鼠体内的平均药-时曲线 Fig. 4 Mean plasma concentration-time profile of silibinin A and silibinin B in rats 表2，表3 水飞蓟宾A和水飞蓟宾B的主要药动学参数 Tab. 2, 3 The main pharmacokinetic parameters of silibinin A and silibinin B 3. 讨论 本实验对正离子和负离子两种电离模式进行了比较，结果显示负离子模式下水飞蓟宾A和水飞蓟宾B的响应值远高于正离子模式，故选负离子模式检测。水飞 -蓟宾和内标的分子离子峰[M-H]分别为m/z 481.1和579.2，由图2可见，水飞蓟宾和内标分别在m/z 300.9和271.1的子离子峰强度最大，故分别选择m/z 481.1? m/z 300.9和m/z 579.2 ? m/z 271.1检测水飞蓟宾和内标。尽管水飞蓟宾A和水飞蓟宾B定量分析的离子反应均为为m/z 481.1? m/z 300.9，但本实验通过比较所选定的色谱条件保证水飞蓟宾A和水飞蓟宾B具有良好的分离度，从而确保了方法具有良好的专属性。 本实验建立了一种简单、快速、灵敏的LC-MS/MS方法，同时测定大鼠血浆中水飞蓟宾A和水飞蓟宾B浓度，并首次报道了水飞蓟宾A和水飞蓟宾B在动物体内的药动学及生物利用度研究数据，为水飞蓟宾及其制剂的深入研究奠定基础。 [参考文献] [1] FLORA K, HAHN M, ROSEN H, et al. 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J Vet Pharmacol Ther, 2007, 30(2): 132-138. [21] WU JW, LIN LC, HUNG SC, et al. Analysis of silibinin in rat plasma - 8 - and bile for hepatobiliary excretion and oral bioavailability application[J]. J Pharm Biomed Anal, 2007, 45(4): 635-641. [22] RICKLING B, HANS B, KRAMARCZYK R, et al. Two high-performance liquid chromatographic assays for the determination of free and total silibinin diastereomers in plasma using column switching with electrochemical detection and reversed-phase chromatography with ultraviolet detection[J]. J Chromato, 2007, 670(2): 267-277. gr B CHCHOHOHOO22CHCHOHOHOO22 OCHOCHHOHOOO33OCHOCHHOHOOO33OOOO OHOHOHOHOHOHOHOHOOOHOHOOOHOH aabb 图1 水飞蓟宾A (a)和水飞蓟宾B (b)的化学结构 Fig. 1 Chemical structures of silibinin A (a) and silibinin B (b) - 9 - 图2 水飞蓟宾A (a)、水飞蓟宾B (b)和柚皮苷(c)的质谱图 Fig. 2 Product ion mass spectrum of silibinin A(a), silibinin B(b) and naringin(c) - 10 - 图3 血浆样品色谱图 a-空白血浆;b-空白血浆加入水飞蓟宾A和内标;c-空白血浆加入水飞蓟宾B和 内标;d-给药后的血浆样品 Fig. 3 Chromatograms of sample in plasma a-blank plasma; b-blank plasma spiked with silibinin A and IS; c-blank plasma spiked with silibinin B and IS; d-plasma sample - 11 - 2400024000300030002400030002400013.3 mg/kg, ig13.3 mg/kg, ig13.3 mg/kg, ig13.3 mg/kg, ig(a)(a)(a)(b)(b)(b)26.6 mg/kg, ig26.6 mg/kg, ig26.6 mg/kg, ig26.6 mg/kg, ig2000020000200002000024002400240053.2 mg/kg, ig53.2 mg/kg, ig53.2 mg/kg, ig53.2 mg/kg, ig16000160001600016000180018001800120001200012000120001200120012008000800080008000 6006006004000400040004000Concentration (ng/mL)Concentration (ng/mL)Concentration (ng/mL)Concentration (ng/mL)Concentration (ng/mL)Concentration (ng/mL) 0000000 03691215036912150369121503691215036912150369121503691215 Time (h)Time (h)Time (h)Time (h)Time (h)Time (h) 30003000300030003000030000300003000014.7 mg/kg, ig14.7 mg/kg, ig14.7 mg/kg, ig14.7 mg/kg, ig(c)(c)(c)(d)(d)(d)29.4 mg/kg, ig29.4 mg/kg, ig29.4 mg/kg, ig29.4 mg/kg, ig24002400240024002400024000240002400058.8 mg/kg, ig58.8 mg/kg, ig58.8 mg/kg, ig58.8 mg/kg, ig 180018001800180018000180001800018000 120001200012000120001200120012001200 6000600060006000600600600600Concentration (ng/mL)Concentration (ng/mL)Concentration (ng/mL)Concentration (ng/mL)Concentration (ng/mL)Concentration (ng/mL) 00000000 0369121503691215036912150369121503691215036912150369121503691215 Time (h)Time (h)Time (h)Time (h)Time (h)Time (h) 图4 水飞蓟宾A和水飞蓟宾B在大鼠体内的平均药-时曲线 a-水飞蓟宾A，静脉注射13.3 mg/kg; b-水飞蓟宾A，灌胃给药13.3, 26.6 and 53.2 mg/kg; c-水飞蓟宾B，静脉注射14.7 mg/kg; d-水飞蓟宾B，灌胃给药14.7, 29.4 and 58.8 mg/kg. Fig. 4 Mean plasma concentration-time profile of silibinin A and silibinin B in rats a-silibinin A, i.v. administration of 13.3 mg/kg; b-silibinin A, i.g. administration of 13.3, 26.6 and 53.2 mg/kg; c-silibinin B, i.v. of 14.7mg/kg; d-Silibinin B, i.g. administration of 14.7, administration 29.4 and 58.8 mg/kg. 精密度与准确度实验结果 表1 Tab. 1 Precision and accuracy of the method Spiked Relative Intra-day Inter-day Conc. error RSD (%) RSD (%) -1(ng?mL) (%) - 12 - Silibinin 20.0 8.3 5.7 3.7 A 500 1.9 2.7 ,1.6 20000 2.1 2.1 ,1.3 20.0 9.1 4.8 5.7 Silibinin 500 5.0 2.1 2.4 B 20000 2.2 0.8 ,2.1 表2 水飞蓟宾A的主要药动学参数(n=5) Tab. 2 The main pharmacokinetic parameters of silibinin A (n=5) i.v. dose i.g. dose Parameters -1-1-1-113.3 mg?kg 13.3 mg?kg 26.6 mg?kg 53.2 mg?kg -1k(1?h) e t(h) 1/2 Cmax 0.18?0.05 0.15?0.07 0.14?0.02 0.16?0.10 -1(ng?mL) 4.11?0.98 5.48?2.23 5.08?0.65 5.73?2.82 T(h) max / 674.3?223.7 1349.4?591.1 2042.5?714.2 AUC 0-t/ 0.20?0.07 0.23?0.09 0.20?0.07 -1(ng?h?mL) 15907.8?3897.9 454.4?152.3 845.9?392.3 1219.5?139.3 16096.4?3941.1 474.4?169.5 871.9?385.1 1272.4?166.6 AUC 0-? 1.05?0.21 1.15?0.27 1.05?0.33 1.11?0.32 -1(ng?h?mL) 14.6?4.1 516.8?178.2 574.0?193.6 708.0?107.0 MRT (h) 5.19?1.82 224.0?68.3 259.6?113.0 334.6?141.8 CL -1(mL?min) V (L) d 表3 水飞蓟宾B的主要药动学参数(n=5) Tab. 3 The main pharmacokinetic parameters of silibinin B (n=5) - 13 - i.v. dose i.g. dose Parameters 14.7 mg/kg 14.7 mg/kg 29.4 mg/kg 58.8 mg/kg -1k(1?h) e t(h) 1/2 Cmax 0.24?0.03 0.18?0.09 0.17?0.02 0.18?0.12 -1(ng?mL) 2.89?0.44 4.56?2.26 4.12?0.58 5.53?4.28 T(h) max / 671.0?274.9 1365.4?733.5 2066.2?848.1 AUC 0-t/ 0.20?0.07 0.23?0.09 0.20?0.07 -1(ng?h?mL) 22432.9?5812.1 432.0?158.2 817.1?454.7 1153.6?205.4 22527.8?5832.8 442.2?165.3 829.4?453.0 1179.4?214.5 AUC 0-? 1.03?0.23 1.02?0.28 0.94?0.28 0.99?0.30 -1(ng?h?mL) 11.6?3.5 616.0?214.5 718.4?314.1 855.2?165.8 MRT (h) 2.92?1.04 225.8?87.1 258.8?124.0 393.0?306.1 CL -1(mL?min) V (L) d - 14 -